The long-range objective of this research proposal is to gain a more detailed understanding of the enzymatic reactions by which glycogen is synthesized and degraded in mammalian (including human) cells and tissues. A large part of the work will be done using cultured human fibroblasts from normal individuals and from patients with various types of glycogenosis. In particular, three enzymes which have already been prepared in this laboratory in a homogeneous state will be studied further to learn more about their structure as proteins and the nature of their active sites. These enzymes are the glycogen debranching enzyme, the glycogen branching enzyme, and the lysosomal alpha- glucosidase. We shall study by enzymic means the structure of the polysaccharide formed from UDPG by the combined synthetic action of glycogen synthase and branching enzyme. The conditions under which glycogen of "normal" average structure can be formed will be studied, and in this way some information about the reason for the existence of specific aggregates of glycogen particles in some cells (liver) and not in others (skeletal muscle) may be obtained. We plan also to study the lysosomal pathway of glycogen catabolism in cultured human fibroblasts in order to gain quantitative information about the extent to which it is normally operative. This will be done by studying the turnover of C14-glycogen under normal culture conditions and also in the presence of disaccharide inhibitors of the lysosomal alpha-glycosidase, which we have recently found to be taken up by such cells thereby inducing glycogen storage as well as prolonging the time during which C14-glycogen persists in the cells. Enzyme studies of glycogen storage disease will be continued particularly with the aim of finding the etiology of those cases of Type VI disease for which there is now no satisfactory uniform explanation.